The Role of Lipid Rafts in Fetal Alcohol Spectrum Disorder

描述(申请人提供)：乙醇是一种已知的人类致畸物质，对公共健康有巨大影响。怀孕期间大量饮酒是导致智力低下的主要已知原因，而更微妙的影响，特别是对发育中的大脑的影响，发生在所有活产婴儿中的1%。最近的证据表明，乙醇对发育中的中枢神经系统的毒性作用的许多已知蛋白质靶标都与脂筏有关。脂筏是质膜上的特殊微域，是蛋白质相互作用的平台。我们以前的工作表明，L1细胞黏附分子(L1)是一种对神经系统正常发育至关重要的蛋白质，它促进轴突生长的能力对乙醇非常敏感。局部破坏脂筏可减少L1介导的轴突生长。我们的初步数据显示，乙醇改变了L1在脂筏中的分布，胆固醇消耗消除了对酒精的敏感性。此外，我们的初步数据表明，乙醇的其他蛋白质靶标在乙醇存在的情况下改变了它们的脂筏分布。我们假设乙醇毒性的一个主要机制是通过改变脂筏-蛋白质相互作用，并且减少毒性的干预措施阻止了这种破坏。这些假说将使用来自大鼠幼鼠和小鼠的小脑颗粒神经元(CGN)进行验证，包括野生型和缺乏L1的小鼠，以及大鼠体内插管模型。在目标1中，我们将通过将缺少L1的CGN与缺乏与脂筏共定位的信号的L1的突变构建体一起导入CGN来证实脂筏-L1的相互作用是轴突生长对乙醇敏感性的基础。预计转基因细胞中的轴突生长对乙醇不敏感。在目标2中，我们将确定L1或脂筏是否是乙醇作用的部位，以及乙醇对L1进出脂筏的动力学的影响。在目标3中，我们将使用在其他实验系统中被证明具有保护性的四种营养物质，以防止继乙醇之后蛋白质分布的变化。这些营养物质的最有效结合将在CGN和活体模型中用L1确定。这项研究的相关性在于，对这些营养素进行简单的饮食控制可以防止乙醇的某些方面的神经毒性，就像补充叶酸和减少神经管缺陷一样。

DESCRIPTION (provided by applicant): Ethanol is a known human teratogen of immense public health impact. Heavy drinking during pregnancy is the leading known cause of mental retardation, while more subtle effects particularly on the developing brain occur in 1% of all liveborns. Recent evidence has shown that many of the known protein targets for ethanol's toxic effects on the developing central nervous system are associated with lipid rafts. Lipid rafts are specialized microdomains of the plasma membrane and serve as platforms for protein-protein interactions. Our prior work has shown that the ability of L1 cell adhesion molecule (L1), a protein critical for the proper development of the nervous system, to promote neurite outgrowth is exquisitely sensitive to ethanol. Localized disruption of lipid rafts reduces L1 mediated neurite outgrowth. Our preliminary data show that ethanol alters the distribution of L1 in lipid rafts, and that cholesterol depletion abolishes ethanol sensitivity. Further, our preliminary data suggests that other protein targets of ethanol change their lipid raft distribution in the presence of ethanol. We hypothesize that a major mechanism of ethanol toxicity is through alteration of lipid raft-protein interactions, and that interventions which reduce toxicity prevent this disruption. These hypotheses will be tested using cerebellar granule neurons (CGN) from rat pups and from mice, both wild type and lacking L1, and a rat in vivo entubation model. In Aim 1, we will confirm that lipid raft-L1 interactions underlie ethanol sensitivity of neurite outgrowth by transfecting CGN lacking L1 with mutant constructs of L1 lacking a signal to colocalize with lipid rafts. Neurite outgrowth in transfected cells is expected to be ethanol insensitive. In Aim 2, we will determine whether L1 or the lipid raft is the site of the ethanol action, and the effect of ethanol on the kinetics of L1 trafficking to and out of the lipid raft compartment. In Aim 3, we will use four nutrients shown to be protective in other experimental systems, to prevent the changes in protein distribution secondary to ethanol. The most effective combination of these nutrients will be determined with L1 in CGN and in the in vivo model. The relevance of this research is that simple dietary manipulations with these nutrients may prevent some aspects of ethanol neurotoxicity much like folic acid supplementation and reduction of neural tube defects.